Hypoallergenic pet food

ABSTRACT

A composition comprising partially hydrolyzed proteins for use as a hypoallergenic pet food, and a process for producing it.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pet food for domestic pets such ascanines including dogs and felines including cats with food allergiesand the like.

2. Description of the Related Art

Food allergies, or food hypersensitivities, commonly afflict householdpets such as dogs and cats. These allergies can cause the pet to exhibitsymptoms such as excessive itching and scratching, diarrhea or othersymptoms which are aggravating both to owner and pet. A housebroken dogwith diarrhea who is indoors has a major problem. If the owner isaround, the dog pesters the owner to get outside. If the owner is notaround the owner may later think the dog is exhibiting undesirablebehavior and needs to be punished.

Domestic pets such as dogs and cats give their owners pleasure and oftencome to be regarded as family members. Accordingly, pet owners alsoexperience distress when their pets develop allergies to their foods.Such food allergies, which are also known as food hypersensitivities,often manifest themselves by itching, swelling, vomiting, diarrhea (andsometimes even bronchoconstriction and anaphylaxis), and can bedifficult to diagnose. It has been estimated that at least 15% of alldogs in the United States suffer from allergies of some type, and that10% of those arise from food hypersensitivities.

Diagnosis is a particular problem for veterinarians, because diagnosisof food hypersensitivity is often difficult and consumes an inordinateamount of veterinarians' time. Despite development of sophisticatedimmunoassay techniques such as radioallergosorbent tests,radioimmunoassay, and enzyme-linked immunoassay, the most reliable wayof diagnosing food allergies is still to replace the pet's usual foodwith another one to which the pet should not be allergic. Traditionallythis has been done by feeding the pet a food it has not eaten before,and to which the pet therefore should not have an allergy.

Simply changing from one protein source to another, however, for severalreasons provides at best a partial solution to a food hypersensitivityproblem. First, the variety of ingredients in commercially available petfoods can make it difficult to find one that includes only food proteinthe pet has not eaten before. Second, intact proteins are intrinsicallyantigenic, and owing to cross-reactivities pets could exhibit allergieseven to unfamiliar food proteins. Third, inflammatory bowel disordersand other pathological conditions such as gastroenteritis can render theintestinal mucosa permeable to proteins larger than can usually crossthe mucosa, and thus these conditions expose gut-associated lymphatictissue to food proteins large enough to be antigenic. As a consequence,pets suffering from such pathologies often quickly become allergic tothe new protein source.

Such reactions to unfamiliar foods can mislead clinicians intoconcluding incorrectly that the pet's problems do not arise from foodhypersensitivity, and thereby delay effective treatment of the problem.In the case of a pet, failure to diagnose the problem can be lifethreatening because the owner may lose patience with repeated“accidents” by the afflicted pet and decide it is time for a new pet.Thus changing from one antigenic protein source to another often failsto solve either diagnostic or long-term maintenance problems arisingfrom food hypersensitivity.

An alternative approach to changing the identity of the food protein isto reduce its antigenicity through heat denaturation. Heat denaturationoften fails, however, because most antigens are heat-resistant.Furthermore, heat denaturation in some cases exposes epitopes hidden inthe native form of the proteins, and therefore can increase rather thandecrease the antigenicity of the heat-denatured protein.

Dialysis and ultrafiltration have also been used to physically removeantigenic proteins such as beta-lactalbumin from cow's milk for humanconsumption. Both are expensive procedures, however, that would becost-prohibitive for pet foods. Hydrolysis of proteins has also beenemployed to mitigate antigenicity in human nutrition and in farm animalhusbandry for calves and pigs. Partially hydrolyzed soy proteins havebeen used as the basis of a milk substitute, and a modified soy flourfor human consumption has been prepared by the action of barleyproteases upon conventional soy flour. Similarly, it has been disclosedto prepare a protein hydrolyzate for human consumption by pancreatichydrolysis of proteins, followed by heat treatment and ultrafiltration.Diets consisting of oligopeptides with four or five amino acid residueshave been prepared for treatment of human Crohn's disease patients.

Such protein treatments, however, as noted above have not been attemptedin pet foods for domestic dogs and cats. The best solution to diagnosisand treatment of food allergies in pets is to have a food source that isintrinsically hypoallergenic. This invention achieves that.

SUMMARY OF THE INVENTION

Successful treatment of food hypersensitivity ideally involves a foodthat is intrinsically hypoallergenic, nutritionally-balanced and thatwhen prepared by hydrolysis of food proteins has a molecular weightrange that in the target species does not engender diarrhea or otherproblems and that reduces the likelihood of an allergic response.

The present invention addresses these needs through providing a pet foodcomprising a proteinaceous component that has undergone sufficienthydrolysis to render it hypoallergenic.

It is thus an object of the present invention to provide anutritionally-balanced pet food that is hypoallergenic.

It is another object of the present invention to provide a food fordiagnosing whether a pet suffers from food hypersensitivity.

It is yet another object of the present invention to provide a food forthe long-term maintenance of a pet.

It is further an object of the invention to provide a process forpreparing such a hypoallergenic pet food.

It is yet another object of the present invention to provide acommercially practicable process for preparing such a hypoallergenic petfood.

The present invention meets the above objects by providing a pet foodcomprising as a proteinaceous component proteins hydrolyzed sufficientlyas to have minimal effect on the immune system, and thus to behypoallergenic.

It further meets the above objects by providing a process for preparingsuch a hypoallergenic pet food by mixing hydrolyzed soy proteins with anedible lipid such as coconut oil to permit extrusion of the pet foodfrom commercially used extruders.

BRIEF DESCRIPTION OF THE DRAWINGS

A better appreciation of the differences in antigenicity and molecularweight of the special formula of the invention may be had by referenceto appended drawings, wherein:

FIG. 1 shows the low level of antigenicity of the hypoallergenicformula.

FIG. 2 shows the higher level of antigenicity of unmodified soy protein.

FIG. 3 shows the molecular weight distribution of the hypoallergenicformula.

FIG. 4 shows the molecular weight distribution of unmodified soyprotein.

It is to be noted, however, that the appended drawings illustrate onlycomparative antigenicity plots and molecular weight distributions forthe purposes of contrasting the pet food formula of the invention fromconventional pet food formulas and that they are therefore not to beconsidered limiting of the scope of the invention, for the invention mayadmit to other formulas with other plots and distributions.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Agglomerating agent” as used herein refers to a composition used toeffect agglomeration of fine powders.

“Allergy” as used herein refers to altered bodily reactivity to asubstance in response to an earlier exposure.

“Allergen” as used herein is a substance that induces an allergy.

“Antigen” as used herein refers to a substance capable of eliciting animmune response in a pet.

“Food hypersensitivity” as used herein is an allergy or otherimmune-mediated adverse reaction to a food fed to a pet.

“Basal mix” as used herein refers to the mixture of nutritionalcomponents that is extruded to produce a pet food.

“Dalton” as used herein conforms to common scientific usage to refer toa single gram atomic weight; thus a kiloDalton, abbreviated kD, is akilogram atomic weight.

“Hypoallergenic” as used herein refers to a composition that when fed toa pet at customary levels that provide adequate nutrition over the pet'slife does not produce a clinically discernible allergic reaction.

“Lipid” as used herein refers to a chemical compound that issubstantially soluble in a non-polar organic solvent such as chloroform,and that includes fats and oils as lipids that are substantially solidand liquid, respectively, at room temperature. “Edible lipids” are thosethat can be eaten in normal dietary quantities without ill effects.

“Pet” refers to a domestic companion animal, and particularly to a dogor cat.

“Proteinaceous component” as used herein refers to that portion of amixture that contains protein or protein fragments.

“Weight average molecular weight” as used herein conforms to commonscientific usage in referring to an average molecular weight calculatedas the mass-weighted average of the molecular weights of the componentsof a mixture.

We have noted that allergies typically result from adverse reactions toproteins, and especially to glycoproteins, that the immune systemrecognizes as foreign. Food proteins, although foreign, usually do notcause allergies because the intestinal mucosa only absorbs proteinsafter they have been extensively hydrolyzed and thereby renderednon-antigenic in the gut. Pathological conditions such as inflammatorybowel disease damage the intestinal mucosa and thereby increase itspermeability to the higher molecular weight, and therefore antigenic,proteins. Exposure of the gut-associated lymphatic tissue to theseantigenic proteins during inflammatory bowel disease is one mechanismthat results in food allergies.

Because of the exquisite sensitivity of the immune system to allergens,we take the approach that it is best to expose the pet to as few proteinsources as possible to avoid the risk of inadvertently exposing the petto adventitious allergens. Thus a hypoallergenic food is ideallyself-contained and nutritionally-balanced, and as such must contain aproteinaceous component. We have concluded that to provoke an immuneresponse a potential allergen has to be of sufficient size to berecognized as foreign by the immune system. Typical food allergens havemolecular weights in the range of 10-70 kD, and they are thus largeenough to provoke an immune response while still small enough to crossthe gut membrane. Pet foods in use heretofore for diagnosis andtreatment of food hypersensitivity have average molecular weights around22 kD, thus making them still in the antigenic range.

In preparing a hypoallergenic pet food the proteinaceous componenttherefore cannot be too large in average size, but it also cannot be toosmall. A proteinaceous component having too low an average molecularweight would consist largely or entirely of highly charged species. Thisis because many amino acid residues in an intact protein bear no charge,but complete hydrolysis renders each residue zwitterionic atphysiological pH. A zwitterion comprises both an anionic and cationicmoiety in the same molecule (the primary amino and carboxylate groups,respectively, in this case), in addition to any charges originallyexisting on the side chains. Hydrolysis therefore creates two newcharges at each amino acid cleaved from a protein. Simple amino acids,because of their high charge, have a strongly bitter taste that isnotoriously unpalatable. Furthermore, ingesting a highly charged specieswould lead to a high osmotic strength in the intestinal tract, whichwould draw water to the gut and thereby provoke diarrhea. Any owner ofan indoor pet knows you do not want that.

Completely hydrolyzed proteins are undesirable for other reasons. It isnot normal for an adult mammal to acquire nutrition from completelyhydrolyzed proteins, particularly as a maintenance diet. The adultmammal's digestive tract is accustomed to digesting proteins, not havingthem supplied already fully digested. In the absence of food proteins tohydrolyze, the pancreatic enzymes secreted into the food slurry in theintestine might attack the intestinal wall. Lack of proteins to digestraises concerns about interfering with the normal functioning of gastrichormones that provide physiological feedback to the digestion process.This particularly troubling because the effect of protein absence ongastrointestinal motor function is not known. Thus it is desirable tosupply nutrition in a diet as close to normal food as is possiblewithout triggering an allergic reaction.

Other considerations also make partial hydrolysis especially desirable.Hydrolysis of proteins is expensive, and hence hydrolyzing the proteinsmore than is necessary to mitigate allergic reactions iscost-prohibitive for a pet food. In addition, simple amino acids tend tobe hygroscopic, that is to absorb water from the air. For commercialpurposes it is especially valuable to be able to distribute the pet foodwithout burdensome precautions to protect it from atmospheric moisture

For all these reasons it is therefore desirable to afford aproteinaceous component with an average molecular weight as high as ispossible without provoking an allergic response in the target species.

Although food allergens can have molecular weights as low as 10 kD, wehave now found that, in dogs, hydrolyzed soy proteins with an averagemolecular weight below 18 kD are sufficiently hypoallergenic to beuseful in the diagnosis and treatment of food allergies. Thus oneembodiment of the present invention includes soy proteins with anaverage molecular weight below 18 kD. A preferred embodiment of thepresent invention includes soy proteins with an average molecular weightof around 12 kD, and with less than 20% of the proteinaceous componenton a weight basis having a molecular weight greater than 20 kD. We havealso found that in a preferred embodiment for cats the hypoallergenicpet food comprises partially hydrolyzed meat-based proteins in theproteinaceous component to increase the acceptance of the pet food bycats.

We have found that to be commercially valuable as a kibble ahypoallergenic pet food should be capable of processing with extrudersand other equipment already in place. We have found that a basal mixtureof partially hydrolyzed soy proteins and corn starch has rheologicalproperties that make it impracticably difficult to process throughextruders now in commercial production use. Corn starch also tends toform explosive aerosols when handled. We have found that agglomeratingthe basal mixture with an agglomerating agent such as coconut oil notonly reduces the explosion hazard, but also modifies the rheologicalproperties of the basal mixture to make it compatible with extrusionthrough the extruders now in use, and thus to be commercially valuable.Simply mixing in the coconut oil into the basal mixture does not producethis benefit. In a preferred embodiment the basal mixture isagglomerated with coconut oil by atomization, and added to the extent ofless than about 5% by weight. In an especially preferred embodiment,coconut oil is atomized onto the basal mixture to the extent of about1-2% by weight. In a still more preferred embodiment, a secondapplication of coconut oils is made before the basal mixture proceeds tothe extruder.

Having prepared a product, a commercial producer needs to be able todistribute it in a marketable form. We have found that a pet foodprepared as described above can become rancid. We have further foundthat if rancidity is a problem its onset can be impeded by packaging thepet food in packaging material that has been impregnated with ananti-oxidant. In an especially preferred embodiment, the container isimpregnated with vitamin E, but a variety of widely used anti-oxidantscan be employed, such as butylated hydroxy toluene (BHT:2,6-di-tert-butyl-4-methylphenol), butylated hydroxy anisole (BHA: amixture of 2- and 3-tert-butyl-4-methoxyphenol),4,4-methylene-bis-2,6-di-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,6-di-tert-butylphenol,ethoxyquin,tetrakis-methylene-(3,5-di-tert-butyl-4-hydroxyhydrocinnamate)methane,tert-butylhydroquinone, and other anti-oxidants well-known in the art tobe effective for protection of oxidizable foodstuffs against oxidation.In an especially preferred embodiment, about 150 ppm of vitamin E isimpregnated in a four-ply bag comprising about 60% high densitypolyethylene and about 40% poly-ethylene.

The hypoallergenic pet food can also be distributed in other formsbesides a kibble. Other useful forms include without limitation aliquid, a gruel, and a powder from which a liquid or a gruel can beprepared. These other useful forms do not require extrusion, and thus donot require agglomeration with an agglomerating agent. Edible lipidswill still be necessary for nutritional purposes, however.

It is apparent that a variety of protein sources other than soy could bepartially hydrolyzed and used as the proteinaceous component of the petfood. Other protein sources could be derived from animal or plantsources. Animal sources of protein comprise mammals and their milk,fowl, amphibians, reptiles, and fish, and include cattle, sheep, pigs,goats, deer, rabbits, horses, kangaroos, catfish, herring, salmon,alligators, iguanas, quail and their eggs, ostriches and their eggs,turkeys and their eggs, chickens and their eggs, and ducks and theireggs.

Plant sources of protein comprise both unicellular and multicellularorganisms, and besides soy beans include algae, yeasts, bacteria, flaxseed, corn, wheat, oats, sorghum, kelp, barley, alfalfa, rye, quinoa,peanuts, rice, and potatoes.

It is also apparent that a variety of other agglomerating agents couldbe used to modify the rheological properties of the pet food basal mixsuch that it can be extruded through conventional extruders. Althoughcoconut oil is preferred, any pet food-compatible agglomerating agentmay be used. Besides coconut oil, other agglomerating agents found to besuitable for this purpose include canola oil, but other edible fats andoils could also be used. Examples of such fats and oils include withoutlimitation almond oil, apricot oil, avocado oil, borage oil, black oil,canola oil, carrot oil, coconut oil, corn oil, crambe oil, currant oil,flax oil, filbert seed oil, grapeseed oil, ground nut oil, hazel nutoil, hop oil, kiwi seed oil, kukui nut oil, macadamia nut oil, mangoseed oil, marigold oil, mustard seed oil, neem oil, olive oil, palm oil,passionflower seed oil, peanut oil, pine nut oil, rapeseed oil, ricebran oil, safflower seed oil, sesame seed oil, soybean oil, sunfloweroil, sweet almond oil, tea seed oil, walnut oil, wheat germ oil, andyucca oil. Animal, fish, and poultry fats can also be used, providedthey have been purified to remove any proteinaceous content that mightbe antigenic.

Besides atomization, other ways of applying agglomerating agent to thebasal mixture include spraying, sonication, or other ways of addingfinely dispersed agglomerating agents such as edible lipids.

Accordingly, it is also apparent from the foregoing that the presentinvention is not limited to the embodiments shown. Other equallyeffective embodiments are contemplated and within the scope of thepresent invention.

EXAMPLES

Preparation of the hypoallergenic pet food. A basal mixture was preparedby mixing together the following ingredients, where PF stands for PetFood grade:

magnesium oxide PF 1.86 lbs corn starch PF 670.63 lbs vitamins superpremium 0.54 lbs vitamin E PF (50% by wt. dl-alpha- 0.12 lbs tocopherolacetate, supplying 500 IU/gm) Calcium carbonate PF 19.60 lbs Hypo ALMineral PX J20 262.49 lbs Dicalcium phosphate 70% human 12.79 lbs gradePF Sodium chloride PF 1.12 lbs Choline chloride 70 PF 2.45 lbs

The vitamin super premium component contains riboflavin (18.47%), groundyellow corn (14.33%), vitamin E (18.02%), biotin (3.7%), vitamin B₁₂(3.255%), calcium pantothenate (14.5%), vitamin D₃ (0.55%), folic acid(1.85%), menadione sodium bisulfate complex (0.4%), niacin (14.72%),pyridoxine hydrochloride (1.50%), thiamine hydrochloride (2.69%).

The Hypo AL Mineral PX J20 component above contains cellulose (21.30lbs), a blend of the food gums guar and gum arabic (5.33 lbs), traceminerals (6.81 lbs) including selenium, dl-methionine (2.40 lbs), zincsulfate (0.04 lbs), isolated partially hydrolyzed soy protein granulesPF (225.43 lbs), and lecithin (feed grade PF; 1.14 lbs). The basalmixture described in the above table was then agglomerated with coconutoil (17.76 lbs) containing a trace of citric acid to facilitatesubsequent processing, extruded through an extruder, and sprayed withcanola oil containing tert-butylhydroquinone as an anti-oxidant.

Molecular weight determinations. Dry samples as well as standards weredissolved in 6 M guanidine hydrochloride (GuHCI)-pH 7.6 phosphate buffercontaining 1 mM ethylenediamine tetraacetic acid (EDTA) and 10 mMdithiothreitol (DTT). The samples were heated at 65° C. for 3 hours toensure the complete dissolution of all proteins in a totally unfolded,random coil state. Under these conditions elution time (volume)correlates with molecular weight.

Gel permeation size exclusion chromatography was performed on a HewlettPackard 1050 chromatography system with a Progel (TSKG3000SWXL) column(resolution 100,000 to 200 daltons for random coil peptides) connectedin series with a GPC-Peptide column (resolution from 10,000 to 250daltons). The columns are purchased from Supelco (Bellefonte, Pa.). Thecolumns were eluted with the GuHCI buffer at a flow rate of 0.47 mL/min,and the eluted proteins detected by absorbance at 280 nm.

The column was calibrated with bovine serum albumin (66 kD), ovalbumin(44 kD), alpha-chymotripsinogen (25.7 kD), myoglobin (17 kD), cytochromec (12.4 kD) and a synthetic hexapeptide (685 daltons). The elution timesof the standards were fitted to the molecular weights by a cubicequation. The molecular weight averages were calculated by integrationfrom the high limit (100 kD) to the low limit (250 daltons).

The average molecular weights were defmed as: weight average molecularweight=MW_(i)=Σ_(i)n_(i)M² _(i)/Σ_(i)n_(i)M_(i), and number averagemolecular weight=MW_(n)=Σ_(i)n_(i)M_(i)/Σ_(i)n_(i), where n_(i)=numberof molecules with molecular weight M_(i) and M_(i)=molecular weight ofthe i^(th) molecule. The polydisparity is the weight average molecularweight divided by the number average molecular weight. A lowpolydispersity indicates narrow molecular weight distribution.

Antigenicity measurements were performed by the method described inMethods of Enzymology, Vol. 118, pp. 742-766.

Sample Preparation. Antigenicity was measured on the soluble portion ofthe sample. For low antigenicity samples, such as those containing onlyhydrolyzed soy protein, the extraction concentration was 15 grams ofsample per 100 mL of buffer. High antigenicity samples, such as those ofnative soy protein, are too viscous at this concentration, and wereextracted at concentrations of 5 grams of sample per 100 mL of buffer.

To the appropriate amount of sample as defined above was added 100 mLcarbonate buffer to a 250 mL beaker. Sample was slowly added to thevortex of carbonate buffer stirring on a magnetic stirrer, and theslurry was stirred until the sample was completely dispersed(approximately 5 minutes). The pH was then adjusted to pH 9.6 with 6 MNaOH, if necessary, and the dispersion was stirred for 30 minutes. ThepH was then readjusted to 9.6, if necessary. The slurry was stirred on amagnetic stirrer for 1 h, and then passed through a portable homogenizertwice before being centrifuged at 12,000×G for 30 minutes at 25-30° C.The supernatant was then decanted and placed in a covered container.Protein content of the supernatant was then measured by the modifiedKjeldahl method, as described below.

Protein determination by the modified Kjeldahl method (Official Methodsof Analysis of the AOAC (Association of Official Analytical Chemists),16th Edition, (1995); 988.05, Locator #4.2.03; 920.87, Locator 32.1.22;and 991.20, Locator # 33.2.11):

One gram of sample was weighed accurately and transferred into an 800 mLKjeldahl flask. Pope Kjeldahl powder (19.8 g) was added to the sample inthe flask, followed by approximately 30 mL of concentrated H₂SO₄. Thesample mixture was digested for approximately one hour, or for about 30minutes longer than the time necessary for the sample solution to clear.The flask was allowed to cool to under 100° C. (estimated) or untilprecipitate started to form, whichever occurred first. The samplesolution was diluted to approximately 400 mL with water, by forcefullyadding the water directly into the acid in the flask to ensure thoroughmixing. A known volume of 0.2000 N H₂SO₄ was placed into a wide mouth500 mL Erlenmeyer flask, followed by five to seven drops of Methyl Redindicator, and the acid solution diluted to approximately 100 mL withdeionized water. The wide mouth Erlenmeyer flask containing the standardacid was placed under the condenser of a distillation unit, with the tipof the condenser submerged in the standard acid.

Approximately 75 mL of 10 N NaOH was added down the neck of the Kjeldahlflask without agitation, and the flask immediately connected to thecondenser. Once connected, the Kjeldahl flask was swirled to mixthoroughly the solution, and the sample solution distilled untilapproximately 200 mL of distillate collected in the Erlenmeyer flaskcontaining the standard acid.

The tip of the condenser tube was rinsed into the Erlenmeyer flaskcontaining the standard acid with deionized water. The Erlenmeyer flaskwas then removed and the distillate titrated to a Methyl Red end point.

Percent nitrogen was calculated as [{(vol. std. acid)×(0.2)×(conc.acid)−(vol. std. base)×(conc. base)−(vol. blank)×(conc.base)}×(0.014007)×(100)]/[(sample weight in grams)], where0.014007=grams per milliequivalent of nitrogen.

Percent nitrogen or percent protein in each sample was calculated as:

% N=[(vol. std. acid−vol. std. base−vol.blank)×(2)×(0.0014007)×(100)]/[sample weight, g],

and

% protein=% N×6.25, where 6.25 is the protein factor.

Sample antigenicity was then determined in a dust-free environment on aseries of sample dilutions, with one positive sample and one negativesample included in each day's testing. A series of 23 halving dilutionsof the sample and the positive reference extract were prepared, andequal volumes of the sample to be tested and carbonate buffer werestirred by a vortex mixer in culture tubes. For each dilution 100microliters were pipetted into each of three wells, with 11 wells perplate left blank of sample and 100 microliters of carbonate buffer addedto them instead. The plate was then covered with a lid and placed in asealed box, where it was allowed to equilibrate for 16-24 hours at 40°C. Any excess sample was then removed by overturning plate and tappingit quickly on a paper towel, after which the plate was washed threetimes with phosphate buffer from a wash bottle, with each washingconsisting of filling the well to volume with buffer. Excess buffer wasthen removed by overturning the plate and tapping it quickly on a papertowel. The wells were then filled to volume, with care being taken notto allow the buffer to overflow into surrounding wells on the plate.After the final wash, the plate was overturned and rapped sharply onclean, dry paper towel.

One microliter of 3.0% rabbit serum was added to each well, and theplate then covered and allowed to incubate for 30 minutes at 37° C.Excess rabbit serum was then removed by aspiration and the plate washedthree times with phosphate buffer as described above. The conjugate wasthen diluted as above and 100 microliters of diluted conjugate added toeach well.

After being covered and equilibrated for 1 hour at 37° C. the excessconjugate was removed and washed three times with phosphate buffer asabove. The substrate was prepared as outlined above and 100 microlitersof prepared substrate added to each well. The plate was covered and thereaction allowed to proceed for 20 minutes at room temperature(approximately 21° C.), in the dark. After 20 minutes, the reaction wasquenched by addition of 100 microliters of 1 M phosphoric acid to eachwell, and the plate was read immediately at 405 nm.

Antigenicity is reported as positive or negative, with the antigenicityvalue determined as follows. From the average of the three absorbancevalues for each sample dilution was subtracted the average blankabsorbance value. If all average absorbance data points fell below 0.100after the average blank value was subtracted, the sample is reported asnegative. If any of the average sample points were 0.100 or greaterafter the average blank value was subtracted, the sample is reported aspositive. Average blank absorbance must be below 0.200 for the resultsof the assay to be valid.

Test on dogs. Six dogs with inflammatory bowel disease were selectedfrom patients presented to the Veterinary Teaching Hospital at theUniversity of California Davis between July and December, 1997. Criteriafor entry into the study included a diagnosis of chronic inflammatorybowel disease based on history, physical examination, fecal flotation,fecal smear, and histological evidence of inflammation as well aswillingness to comply with the guidelines of the study. Serum B₁₂,folate, and trypsin-like immunoreactivity were also measured at thebeginning of the study.

Gastroduodenoscopy and biopsy were performed prior to and after 8 to 14weeks of dietary therapy. All owners completed a questionnaire every twoweeks during the 10 week study. The questionnaire entailed determinationof the dog's appetite, body weight, coat condition, frequency ofdefecation, and stool quality. Stool quality was assessed according to ascore of 0 to 100 with 0 being very watery stool and 100 being very firmstool. Illustrated guidelines were provided to the clients to aidconsistency in scoring.

Initial therapy for all dogs was restricted to the feeding of the testdiet. All dogs were fed the test diet twice daily, with total intakecalculated to meet energy requirements (132*(body weight)_(kg) ^(0.75)).No other medical therapy was used in the initial management of the dogs,and considerable emphasis was placed on “educating” owners on theimportance of feeding only the diet with absolutely no treats. Patientsnot showing adequate improvement in clinical signs after 2 to 4 weeks ondiet alone had appropriate medical therapy added to the dietary regime.

Intestinal biopsies were examined in a blinded fashion by a singlepathologist using a numeric grading scheme. In this scheme points wereassigned to each of the following changes in the specimens: one pointeach for villus blunting, villus fusion, minimal to mild inflammationwithin the lamina propria and/or submucosa, mild infiltration oflymphocytes into the mucosal epithelium, erosion or necrosis,lymphangiectasia of the lacteals, fibrosis, crypt or gland ectasia; twopoints each for moderate inflammation within the lamina propria and/orsubmucosa, or moderate infiltration of lymphocytes into the mucosalepithelium; three points each for severe infiltration of the laminapropria and/or submucosa, or severe infiltration of lymphocytes into themucosal epithelium. The degree of inflammation was based on the relativepercentage of inflammation (above a subjective determination of residentlymphoplasmacytic populations to the combined amount of connectivetissue and smooth muscle in the lamina propria. Inflammation wascategorized as: mild if the sample contained less than 40% inflammatorycells, moderate if the sampled contained 40 to 60% inflammatory cells,and severe if the sample contained over 60% inflammatory cells. Gradingwas based on the total score of abnormal findings in each section. Mildpathology was defined as a total score less than 2; a score between 3and 6 was defined as moderate pathology; and severe pathology wasdefined as a score greater than 7.

Data were analyzed for normality of distribution and the treatmenteffect was analyzed by paired t-tests, using Sigmastat (SigmaStat 2.0,Jandel Scientific, San Rafael Calif.). Test criteria were set atα=0.050, β=0.750.

Six different breeds were evaluated with a mean age of 3.3±1.2 years.The duration of vomiting or diarrhea ranged from 3 to 18 months. Fivedogs had been previously refractory to a variety of commercial andhome-cooked elimination diets, and three dogs (subjects 1, 2 and 6) hadfailed to respond to concurrent medical therapy with metronidazole,metoclopramide, famotidine, amoxicillin, or diphenhydramine.

Fecal scores, based upon a standardized subjective comparison, improvedsignificantly, from a mean of 42.5 before therapy to 91.7 after therapy.Dietary therapy alone provided adequate clinical improvement in fourdogs. Additional medical therapy was required in two patients. A dogwith inflammatory bowel disease and concurrent exocrine pancreaticinsufficiency required pancreatic enzyme replacement, and metronidazolebefore its stools improved. A patient that presented with vomitingassociated with lymphoplasmacytic gastritis showed a moderate reductionin the frequency of vomiting episodes following dietary interventionalone, and complete resolution of vomiting following dietary therapy andmetoclopramide. Four dogs showed histologic improvement in their gastricbiopsies, although the overall effect of treatment did not reachstatistical significance. Significant histologic improvement occurred induodenal biopsies following therapy.

TABLE 1 Summary of signalment and response to dietary therapy in dogswith chronic inflammatory bowel disease Signal- BCS Clinical out-Additional Chg BW Subject ment History (9 pt) come therapy (kg) 1 2.5 yrM 6 mo history vom- 3 Complete None +6.6 Coon- iting and diarrhearesolution hound (lg and sm bowel), wt loss (6 kg) 2 9 y F 3 mo historyvom- 6 Complete None +6.5 Collie iting & diarrhea resolution (sm bowel),wt loss (3 kg) 3 2 yr FS 18 mo history of 4 Moderate im- Metoclo- −0.7Puli intermittant vom- provement pramide iting 4 1.5 yr 18 mo history 3Complete None 0 FS vomiting & diar- resolution Rhod. rhea (lg & sm Ridgebowel), wt loss (4 kg) 5 3 Yr F 3 mo history diar- 3 Moderate im-Pancrea- 0 German rhea (sm bowel), wt provement zyme, Shep- loss (5 kg).Con- Predni- herd current EPI sone, Met- ronidazole 6 1.5 yr 6 mohistory vom- 7 Moderate im- None 0 MC iting & diarrhea (lg provementDalma- bowel) tion BW = body weight; BCS = body condition score, asdefined in D. P. Laflamme, “Body Condition Scoring and WeightMaintenance,” Proc. N. Am. Vet. Conf. Jan. 16-21, 1993, Orlando, FL, pp290-291; D. P. Laflamme, R. D. Kealy, and D. A. Schmidt, “Estimation ofBody Fat by Body Condition Score,” J. Vet. Int. Med. 1994 8:154; D. P.Laflamme, G. Kuhlman, D. F. Lawler, R. D. Kealy, and D. A. Schmidt,“Obesity Management # in Dogs,” J. Vet Clin. Nutr. 1994 1:59-65; EPI =exocrine pancreatic insufficiency; M = male; F = female; FS = female(spayed); MC = male, castrated.

The above examples are provided by way of illustrating the invention,and not to serve as limitations to it.

What is claimed is:
 1. A solid hypoallergenic pet food providing amaintenance diet for pets comprising a component that contains proteinor protein fragments wherein all of said component is partiallyhydrolyzed such that the average molecular weight of said component isabout 12 kD and at least a portion of but no more that 20% of the totalprotein on a weight basis has a molecular weight greater than 20 kD. 2.The pet food of claim 1, wherein the protein component is hydrolyzed bya method selected from the group consisting of acidic hydrolysis,alkaline hydrolysis, and enzymatic hydrolysis.
 3. The pet food of claim1, wherein the protein component is chosen from the group consisting ofplant sources and animal sources.
 4. The pet food of claim 3, whereinthe plant source is chosen from the group consisting of soy beans,algae, yeast, bacteria, flaxseed, corn, wheat, oats, sorghum, kelp,barley, alfalfa, rye, quinoa, peanuts, rice, and potatoes.
 5. The petfood of claim 3, wherein the animal source is chosen from the groupconsisting of mammals and their milk, fowl, and fish.
 6. The pet food ofclaim 5, wherein the animal source is chosen from the group consistingof cattle, sheep, pigs, goats, deer, rabbits, horses, kangaroos,catfish, herring, salmon, ostriches and their eggs, turkeys and theireggs, chickens and their eggs, and ducks and their eggs.
 7. The pet foodof claim 1, wherein the pet food is in a form chosen from the groupconsisting of a kibble, a gruel, and a powder from which a gruel can beprepared.
 8. A solid hypoallergenic pet food providing a maintenancediet for pets comprising a component that contains protein or proteinfragments wherein all of said component is partially hydrolyzed suchthat the average molecular weight of said component is below 18 kD and aleast a portion of but no more than 20% of the total protein on a weightbasis has a molecular weight greater than 20 kD.
 9. The pet food ofclaim 8, wherein the protein component is hydrolyzed by a methodselected from the group consisting of acidic hydrolysis, alkalinehydrolysis and enzymatic hydrolysis.
 10. The pet food of claim 8 whereinthe protein component is chosen from the group consisting of plantsources and animal sources.
 11. The pet food of claim 10 wherein theplant source is chosen from the group consisting of soy beans, algae,yeast, bacteria, flaxseed, corn, wheat, oats, sorghum, barley, alfalfa,rye, quinoa, peanuts, rice, and potatoes.
 12. The pet food of claim 10,wherein the animal source is chosen from the group consisting of mammalsand their milk, fowl and fish.
 13. The pet food of claim 8, wherein thepet food is in a form chosen from the group consisting of a kibble, agruel and a powder from which a gruel can be prepared.